Replicating RNA vaccination elicits an unexpected immune response that efficiently protects mice against lethal Crimean-Congo hemorrhagic fever virus challenge

Abstract

Background: Crimean-Congo hemorrhagic fever virus is the cause of a severe hemorrhagic fever with cases reported throughout a wide-geographic region. Spread by the bite of infected ticks, contact with infected livestock or in the health care setting, disease begins as a non-specific febrile illness that can rapidly progress to hemorrhagic manifestations. Currently, there are no approved vaccines and antivirals such as ribavirin have unclear efficacy. Thus treatment is mostly limited to supportive care.

Methods: In this report we evaluated an alphavirus-based replicon RNA vaccine expressing either the CCHFV nucleoprotein or glycoprotein precursor in a stringent, heterologous lethal challenge mouse model.

Findings: Vaccination with the RNA expressing the nucleoprotein alone could confer complete protection against clinical disease, but vaccination with a combination of both the nucleoprotein and glycoprotein precursor afforded robust protection against disease and viral replication. Protection from lethal challenge required as little as a single immunization with 100ng of RNA. Unexpectedly, analysis of the immune responses elicited by the vaccine components showed that vaccination resulted in antibodies against the internal viral nucleoprotein and cellular immunity against the virion-exposed glycoproteins.

Interpretation: Cumulatively this vaccine conferred robust protection against Crimean-Congo hemorrhagic fever virus and supports continued development of this vaccine candidate.

Funding: This research was supported by the Intramural Research Program of the NIAID/NIH and HDT Bio.

Keywords: CCHFV; Crimean-Congo hemorrhagic fever; Mouse; RNA vaccine; Vaccine.

Figure 1

repRNA vaccination elicits humoral and cellular immunity to CCHFV. WT C57BL6/J mice were given (a) prime boost vaccinations at days -56 and -28 relative to CCHFV challenge on day 0. On day 0 groups of four mice vaccinated with 2.5μg (sham, repNP, repGPC) or 5μg (repNP + repGPC) of RNA were evaluated for immune responses to CCHFV. CCHFV-specific antibody was measured by whole-virion ELISA for total IgG (b) or specific isotypes (c). Dashed line indicates background absorbance of wells that received no serum. Serum neutralization activity was measured by a microneutralization assay against infectious CCHFV strain Hoti (d). Dashed line indicates limit of detection and statistical significance calculated using a one-way ANOVA with Dunnett's multiple comparison test. CCHFV-specific T-cell responses were measured by IFNγ ELISpot (e – g). Cumulative SFCs against peptide pools spanning the entire NP or GPC, the mitogen concanavalin a (CA) or DMSO vehicle alone (veh) are shown. Statistical comparisons calculated using a two-way ANOVA with Dunnett's multiple comparison test (c – e) Heat maps showing the distribution of measured IFNγ SFCs against NP (e) or GPC (f) peptide pools. ns P > 0.05, *** P < 0.001. (b – e) Data shown as mean plus standard deviation.

Figure 2

repNP vaccination confers significant protection against CCHFV. Groups of WT mice given indicated prime-boost vaccinations were treated with MAR1-5A3 to blockade type I IFN and infected with CCHFV strain UG3010. Mice were weighed daily (a), monitored for survival (b) and body temperature continuously monitored via telemetry system (c). N = 8 mice per group. Statistical comparisons were calculated using a two-way ANOVA with Dunnett's multiple comparison test (a) or Log-Rank test with Bonferonni's correction for multiple comparisons (b). Statistical significance compared to sham vaccinated animals is shown with symbols: * (repNP), # (repGPC) and + (repNP + repGPC). Viral loads in indicated tissues at day 5 p.i was quantified by qRT-PCR. Dashed line indicates limit of detection (d). N = 6 mice per group Statistical comparisons calculated using a one-way ANOVA with Tukey's multiple comparison test. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (a, c, d) Data shown as mean plus standard deviation.

Figure 3

repNP vaccination protects against liver pathology in CCHFV infected mice. Groups of WT mice given indicated prime-boost vaccinations were treated with MAR1-5A3 to blockade type I IFN and infected with CCHFV strain UG3010. On day +5 p.i. mice were euthanized, liver collected and formalin fixed. Sections were H&E stained or probed for presence of viral antigen via immunohistochemistry (IHC). Representative images for each group are shown at 100x or 400x (inset) magnification and scale bars indicate 100μm or 20μm respectively. Complete findings are provided in Supplemental Table 1.

Figure 4

Single-immunization with repRNA induces humoral and cellular immunity to CCHFV. Groups of 4 WT mice were vaccinated with indicated cumulative doses of repNP + repGPC RNA in a prime-boost regimen as before or in a prime-only regimen. Four weeks after last vaccination, vaccine-induced immune responses to vaccination were measured by ELISA (a) or IFNγ ELISpot (b). CCHFV-specific IgG responses were measured by whole virion ELISA (a). Dashed line indicates background absorbance of wells receiving no serum. Statistical comparisons calculated using a two-way ANOVA using Tukey's multiple comparisons test. The summary P value of each vaccine group individually compared against sham-vaccinated animals is also shown. (b) CCHFV-specific T-cell responses were measured by IFNγ ELISpot and cumulative SFCs against the NP or GPC peptide pools is shown. Indicated statistical comparisons calculated using a two-way ANOVA with Tukey's multiple comparisons test. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001. Data shown as mean plus standard deviation.

Figure 5

Prime-only vaccination protects against CCHFV challenge. Groups of WT mice receiving indicated vaccinations were treated with MAR1-5A3 to abolish type I IFN signaling and challenged with CCHFV strain UG3010. Mice were weighed daily (a), monitored for survival (b) and body temperature monitored continuously via telemetry system (c). N = 8 mice per group except for sham-vaccinated telemetry data where N = 6. Statistical comparison to sham-vaccinated mice performed using a two-way ANOVA with Dunnett's multiple comparisons test or log-rank test with Bonferroni's correction for multiple comparisons (b). At day 5 p.i., viral loads in indicated tissues were quantified by qRT-PCR (d) or TCID₅₀ assay (e) and statistical comparison between sham vaccinated mice and repRNA-vaccinated mice calculated using one-way ANOVA with Dunnett's multiple comparisons test. Statistical comparisons between sham-vaccinated and every repNP-vaccinated groups were significant. N = 6 per group. ns P >0.05, * P < 0.05, ** P < 0.01, *** P <0.001, **** P < 0.0001. (a, c, d, e) Data shown as mean plus standard deviation.

Figure 6

Humoral immunity is required for protection from CCHFV challenge. Groups of WT or B-cell deficient μMT mice were vaccinated with 1μg of repNP + repGPC RNA or sham vaccinated in a prime-only vaccination four weeks prior to CCHFV challenge (a). On days -5 and -2 relative to CCHFV challenge, groups of repNP + repGPC vaccinated mice were treated with an isotype control or antibodies to deplete CD4 T-cells (α-CD4), CD8 T-cells (α-CD8) or both (α-CD4/ α-CD8) (a). Mice were weighed daily (b), monitored for survival (c) and body temperature monitored continuously using telemetry system (d). N = 16 for sham and 6-8 mice per other groups. Statistical comparisons performed using a two-way ANOVA with Dunnett's multiple comparisons test (b) or log-rank test with Bonferroni's correction for multiple comparisons (c). At day 5 p.i., viral loads in indicated tissues were quantified by qRT-PCR (e) and indicated statistical comparisons calculated using one-way ANOVA with Dunnett's multiple comparisons test. N = 6 per group. (b, d, e) Data shown as mean plus standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.